1. Field of the Invention
This invention relates to an autoradiographic genescreening method.
2. Description of Prior Arts
In molecular biology which has been rapidly developed in recent years, it is essential to obtain genetic information on organisms so as to make the function of the organisms or the mechanism of replication clear. For this purpose, it is required to judge whether a gene having specific genetic information does exist in the tissues of organisms or in substances originating from the organisms or not, and to screen and recover the specific gene. This process is termed "gene-screening". Further, it is essential in genetic engineering that the existence of recombinant DNA is confirmed and the recombinant DNA is screened and recovered.
The gene-screening serves as an important means for identifying the corresponding gene in the genetic diagnosis of hereditary diseases. For example, rapid diagnosis for such a disease as cancer at molecular level is eagerly desired. It is also desired to establish a method for diagnosing a pregnant woman on whether a fetus in her body has inborn abnormality or not. It is significantly important that the diagnosis of these diseases can be made by detecting variance in the level of genes.
Accordingly, the detection or screening of genes having specific genetic information is of great value in the above-mentioned various fields. However, the ratio of the specific gene to the whole genomes is very low so that the practical detection or screening of the specific gene is very difficult.
As methods for screening genes, various attempts have been made. As typical methods, there can be mentioned: a chemical analysis method such as column chromatography or electrophoresis utilizing a difference in molecular weights of genes, chemical characteristics thereof, etc.; a biological method which comprises screening the specific genes by utilizing a difference in manifestation character against drug resistance, enzyme activity, etc.; and a probe method which utilizes the ability capable of forming a hybrid between complementary DNAs and RNAs (that is called "hybridization method").
Among these screening methods, the chemical analysis method is hardly employed in practice, because the amount of gene is very small and further the proportion of the specific gene to the whole genomes is very low so that it is very difficult to selectively detect or separate the specific gene. The biological method has a disadvantage in that manifestation of function related to the specific gene is indispensable to the method and the specific gene itself is not always active. As compared with these two methods, the probe method has advantages in that hybridization occurs very selectively and that the procedure is very simple.
As the typical gene-screening method utilizing a hybridization process according to the probe method, there can be mentioned the Southern blotting method. This method can be carried out in the following manner.
In the first place, a large amount of DNAs (or DNA fragments) containing the specific gene are resolved (or developed) on a medium, for example, by means of electrophoresis.
In the second place, double-stranded DNAs on the medium are denatured to form single-stranded DNAs. At least a portion of the denatured DNAs are then transferred to a transfer support (i.e., transfer medium) such as a nitrocellulose filter and fixed thereonto. A hybridization treatment is then carried out on the resulting transfer support.
In carrying out the hybridization treatment, DNA or RNA which is complementary to DNA containing the specific gene is radioactively labeled to prepare a probe. The radioactively labeled DNA or RNA is then hybridized with the denatured DNA on the transfer support. Thus, only a hybrid of DNA containing the specific gene with the radioactively labeled DNA or RNA is formed on the transfer support and it is at the same time radioactively labeled. Namely, the denatured DNA containing the specific gene is hybridized with the radioactively labeled DNA or RNA by heat treatment, and the renaturation of doublestranded DNA or the formation of a DNA-RNA hybrid takes place on the transfer support.
After the hybridization treatment is complete, the transfer support is subjected to autoradiography for detecting the DNA containing the specific gene. Further, after the DNA containing the specific gene is identified, only the DNA can be selectively isolated from the medium used for resolution.
Accordingly, through the above-stated gene-screening procedure (utilizing a hybridization process) according to the Southern blotting method (which is sometimes called Southern transfer method), DNA containing the specific gene can be detected and identified.
As another method for screening genes utilizing a hybridization process, there can be mentioned a screening method according to Northern blotting method.
In the Northern blotting mehod (which is sometimes called Northern transfer method), specimens are RNAs or RNA fragments and a hybridization procedure is carried out in a similar manner to that described above, using radioactively labeled DNA as a radioactively labeled probe.
The above-summerized gene-screening methods utilizing the Southern blotting method or Northern blotting method are described in more detail in the following texts.
METHOD IN ENZYMOLOGY, Vol. 68, pp. 152-176, pp. 220-242, edited by Ray Wu, ACADEMIC PRESS, New York, 1979.
PROTEIN, NUCLEIC ACID & ENZYME (in Japanese), Vol. 26, No. 4, pp. 584-590 (1981)
In carrying out the conventional autoradiography employed for the gene-screening, a radiographic film such as a high-speed X-ray film is combined in layers with a transfer support retaining a captured radioactively labeled probe for a given time so that the film is exposed to the radiation from the transfer support. A radiographic intensifying screen is generally employed to enhance the detection sensitivity of autoradiography. Such autoradiography is described, for example, in the following text: Method in Biochemical Experiment, Vol. 6, Method in Tracer Experiment I, pp. 271-289, "8. Autoradiography" by Toru Sueyoshi & Akiyo Shigematsu (Tokyo Kagaku Dozin Ltd., 1977).
Therefore, the autoradiography is an important means for detecting the specific gene in the gene-screening method. Further, it can be said that the autoradiography is a very useful means, since the identification of the specific gene as well as the isolation and the purification of said gene can be done according to the obtained two-dimensional information on the location of said gene. Nevertheless, such useful autoradiography is not free from several drawbacks in the practical use when applied to the gene-screening method utilizing the hybridization mentioned above.
As described above, in the conventional autoradiography, a transfer support containing a radioactively labeled substance is brought into contact in layers with a radiographic film such as a high-speed X-ray film for a given time so that the film is exposed to the radiation and then a visible image indicating the position of the radioactive substance is obtained.
The primary drawback resides in that the exposure operation requires a long period of time. The exposure operation in the conventional autoradiographic screening is usually carried out for several days, and requires at least several tens of hours even when a radiographic intensifying screen is employed. This is because the amount of nucleic acid such as DNA fixed to the transfer support is small and the radioactively labeled substance (radioactively labeled probe) is generally a nucleic acid partially labeled with .sup.32 P, etc. so that intense radioactivity is not imparted thereto.
The second drawback resides in that the exposure operation should be carried out usually at a low temperature, for example, a temperature in the range of 0.degree. C. to -80.degree. C. This is because a latent image in silver salt of the film formed by exposure to a radiation or light emission, tends to fade at a relatively high temperature such as room temperature, and the so degraded latent image can be no longer developed to give a readable image. Further, the silver salt is easily fogged chemically through migration of deleterious ingredients from the hybridization-treated transfer support to the silver salt layer at such a high temperature. Another reason resides in that the silver salt forms a latent image with difficulty at a relatively high temperature such as room temperature even in the case of utilizing an intensifying screen, because the screen gives an emission of low intensity.
The third drawback resides in that the exposure ought to be carried out in a dry state to prevent the radiographic film from wetting and being fogged. Generally, the exposure is done after the transfer support is dried, or after the support is enclosed in a synthetic resin wrapping film, etc.
When the image obtained by the autoradiography is fogged as described above, the hydridized nucleic acid is hardly located on the obtained image and hence, the result of screening is made remarkably unfavorable.
For these reasons, the operation involved in the conventional autoradiography is complicated, whereby the gene-screening procedure is made complicated as a whole.
Other drawbacks of the conventional autoradiographic gene-screening method are given below.
The photosensitive silver salt of the radiographic film is readily influenced by physical irritation and the radiographic film easily produces fogging under application of physical pressure caused by the contact of the film with the hands of operators or the instrument in the exposure operation. Such unfavorable phenomenon also causes lowering in accuracy of the gene-screening. In order to avoid the occurrence of physical fogging on the radiographic film, high skill and caution must be taken in the handling of the film and hence, the screening operation is further complicated.
The exposure operation in the conventional autoradiographic gene-screening method is conducted over a long period of time as described above so that it is unavoidable that the radiations from natural origin and radioactive impurities incorporated in the transfer support in addition to the radioactively labeled substance take part in the exposure of the radiographic film. Thus, the accuracy of the locational information on the labeled substance is lowered. In order to eliminate such interference and to set appropriate exposure conditions, parallel experiments using control samples are generally carried out to find out proper exposure time, but such experiments have disadvantages in that the number of experiments is increased because such parallel experiments and preliminary experiments for ascertaining appropriate exposure time are involved and hence, the operation is made complicated and less economical as a whole.
The operation of collecting the specific gene is performed in such a manner that the medium carrying nucleic acids (sample) resolved thereon is aligned with the radiographic film on which the autoradiograph of the transfer support is visualized to allow the gene of nucleic acid corresponding to positive-signals indicating the presence of the radioactively labeled substance to be identified and collected. Therefore, if the visualized autoradiograph does not have a satisfactory image indicating locational information owing to the improper conditions for the exposure operation of the transfer support, the accuracy of the gene-screening is lowered. Otherwise, the screening becomes impossible in some cases and accordingly the number of the screening operation involved necessarily increases.
Another disadvantage encountered in the conventional screening method utilizing hybridization technique and exposure to high-speed X-ray film is the difference of the size between the autoradiographed image and the gel support medium used to resolve DNA fragments. This difference of size is caused by shrinking of a transfer support during the transfer and fixing procedure. Therefore, great care should be taken on the identification and collection process.
The gene-screening method utilizing Southern blotting method shows prominently high sensitivity. It is required to detect a single gene from DNAs of human genome in the diagnosis of hereditary disease, etc. in the case that this method is utilized. Accordingly, it is desired to keep the accuracy of the image of autoradiograph from being lowered by the above-described phenomena and to enhance the accuracy of the gene-screening.